B-cell depletion revives the B lineage in aged mice. Old B10D2 (20 months) WT mice (n = 5) were subjected to 3 rounds of B-cell depletion. In each round, mice were first injected intraperitoneally (day 0) with a mixture of monoclonal antibodies specific to B220 (150 μg/mouse), CD19 (150 μg/mouse), and CD22 (150 μg/mouse), followed by a second intraperitoneal injection of rat anti-mouse κ monoclonal antibodies (150μg/mouse) 48 hours later (more details on the effectiveness and the specificity of the depletion is detailed shown in supplemental Figure 2). Blood samples were collected at various times after depletion and analyzed for B-cell frequency (% B220⁺/CD19⁺ cells). The second and the third rounds were administered to each mouse only after more than 80% of the initial B-cell numbers had been restored. (A) Peripheral blood staining of representative young and old mice. Before treatment, old mice were prescreened to have less than 5% AA4.1⁺ B cells in peripheral blood as an indication for an “old-like” B-cell phenotype (see also supplemental Figure 4). (B) Kinetics of B-cell reconstitution after depletion. The time course of B-cell return after each depletion cycle is shown for 3 (of 5) individual mice. The histogram in each plot corresponds to the B-cell frequency in peripheral blood before the first depletion. (C) FACS analysis of the B lineage cells in the BM of mice subjected to 3 rounds of depletion. Mice were analyzed 10 to 14 days after restoring 100% of the B-cell counts in peripheral blood (100-120 days after first depletion was administered). BM cells were stained and analyzed by FACS with a lymphocyte gate as defined by light scatter. The IgM versus AA4.1 plot (bottom) was gated for B220⁺ cells. Representative plots of 5 mice are shown. (D-E) Absolute numbers of B220⁺ cells in BM and spleen (D), and of specific B-cell populations in the BM (E). Values are mean ± SD (× 10⁶). The B-cell numbers were calculated based on the total number of nucleated cells purified from 2 femurs and 2 tibias (n = 5 mice in each group). *Significant difference (P < .05). (F) FACS analysis of the B lineage cells in the spleen of mice subjected to 3 rounds of depletion. Spleen cell analysis for B220 and PanCD45 was conducted on gated IgM⁺ cells. Gates pointed by arrows are for the PanCD45⁺/B220⁺ and the PanCD45⁺/B220^lo populations. Representative plots of 5 mice are shown.